Laser-induced fluorescence analysis device and separation apparatus comprising same

ABSTRACT

The invention concerns a laser-induced fluorescence analysis device ( 1 ), comprising a tube ( 2 ) including a channel, means projecting, an excitation light ( 13 ), optical collecting means ( 7 ), optical measurement means ( 8 ) and a processing means ( 9 ) for producing an analysis result, said collecting means being mechanically coupled to one end of the channel, the tube being integrally made of a material with refractive index either lower than that of water, or higher than both that of water and that of ambient air so as to guide the fluorescence light along said channel in said channel and/or its wall up to the collecting means, which is designed to collect the fluorescence light propagated along the longitudinal direction of the channel.

[0001] The present invention relates to a laser-induced fluorescenceanalysis device for producing fluorescence light from dissolvedsubstances and for detecting this light for the purpose of chemical andbiochemical analysis. The present invention also relates to a separationapparatus that includes such a device.

[0002] It is known to make laser-induced fluorescence measurements inorder to identify and assay substances present in a solution,particularly in trace form. Such measurements have many applications,for example in biochemistry.

[0003] Document U.S. Pat. No. 6,011,882 discloses a chemical detector inwhich a tube made of a polymer known under the registered trademarkTeflon A.F. 2400® is filled with an indicating reactant in liquid phase.The tube is exposed to a substance in vapor phase that it is desired todetect, which substance migrates through the wall of the tube thanks tothe gas permeability of the material of which it is formed. The changeof an optical characteristic of the indicating reactor upon contact withthe substance in question is then detected. Described among the variousdetection techniques envisioned in that document are the detection ofabsorption and the detection of laser-induced fluorescence, whichtechniques use a light source that axially illuminates the inside of thetube from one of its ends and an optical fiber that axially collects thelight at the other end of the tube in order to convey it to an analyzer.However, this arrangement is clearly more favorable to absorptiondetection since the waveguide properties of the Teflon tube conduct thelight injected at the first end to the analyzer, so that, in the case ofa fluorescence measurement, the analyzer receives a strong injectedlight signal superimposed on the fluorescence light signal to bedetected, which very greatly degrades the sensitivity of thefluorescence measurement. Furthermore, this detector is not designed towork in conjunction with a separation system.

[0004] It is known that the sensitivity of fluorescence detection isincreased by preventing the collection, at the same time as thefluorescence light, stray light and/or reflected or scattered excitationlight. To avoid collecting excitation light, the document AnalyticalChemistry, Vol. 72, No. 15, pp 3423-3430 (2000) and the document WO00/04371 propose a capillary electrophoresis arrangement that uses asilica capillary coated on the outside with a layer of polymer having arefractive index lower than that of a separation medium filling theinside of the capillary, in which arrangement the capillary isilluminated in an orthogonal geometry, the direction of propagation ofthe excitation light being transverse to the capillary, whereas thefluorescence light is collected in the axial direction of the capillary.According to that document, the light that is emitted or scattered nearthe outer surface of the capillary propagates spirally along the entirelength of the capillary, close to its outer surface, whereas the lightemitted at the center of the capillary, such as the laser-inducedfluorescence, statistically leaves the capillary near its center,thereby allowing the stray light at the detector to undergo spatialfiltering. However, these documents remain silent about combining theanalysis tube with a separation column or a discharge pipe of theseparation system.

[0005] Document WO 96/15438 discloses another fluorescence detector inwhich the fluorescence light produced in an excitation region of aquartz tube is guided some distance away from the excitation regionusing a collector sleeve which must have a refractive index greater thanthat of the specimen occupying the inside of the tube and which is madeof a material having essentially the same refractive index as the tube,said sleeve being fastened to the tube by melting, adhesive bonding, ormolding. However, this detector has drawbacks insofar as the collectorsleeve entails an additional cost and the interface between thecollector sleeve and the tube ensuring optical contact is difficult toimplement and/or is of limited effectiveness over time.

[0006] International application WO 00/60342 discloses a laser-inducedfluorescence analysis device of the type comprising:

[0007] a tube having a channel capable of containing a solutioncomprising at least one substance able to undergo a laser-inducedfluorescence reaction;

[0008] a projection means capable of projecting an excitation light beamon a portion of said channel in a direction making an angle of greaterthan 60° to a longitudinal direction of said channel, said excitationlight being capable of inducing a fluorescence reaction in saidsubstance or one of said substances;

[0009] an optical collecting means placed so as to collect fluorescencelight from said channel;

[0010] an optical measurement means coupled to said collecting means soas to be able to measure said collected fluorescence light; and

[0011] a processing means capable of processing a measurement signaltransmitted by said measurement means in order to produce a result ofthe analysis of said solution.

[0012] More precisely, this known device comprises a capillary in whicha solution to be analyzed, comprising a solute that becomes fluorescentunder light excitation at a certain wavelength, is conveyed from aseparation system, which may be a high-performance liquid chromatography(HPLC), a micro-high-performance liquid chromatography (μ-HPLC) orcapillary electrophoresis (CE) system. A laser illuminates,perpendicular to the direction of the capillary, an analysis cell insidethe capillary, the wavelength of the laser being chosen in order toexcite the fluorescence of said solute. A ball-shaped lens is alsoprovided for collecting fluorescence light from the analysis cell, aphotomultiplier tube for measuring the collected fluorescence light anda means for analyzing the measurement signals produced by the photomultiplier tube in order to give a result of the analysis.

[0013] However, in that device the optical path of the excitation lightand the optical path of the fluorescence light are partly collinear, sothat a spectral filtering means, in the form of a dichroic mirror, isneeded to separate them. However, such systems collecting fluorescenceonly in the excitation direction can collect only a small portion ofthis fluorescence emitted isotropically in space.

[0014] The object of the present invention is to provide a highlysensitive laser-induced fluorescence analysis device that can becombined with or included in a high-performance liquid chromatography,micro-high performance liquid chromatography, or capillaryelectrophoresis system and does not have the aforementioned drawbacks orsome of them. The object of the invention is also to provide aseparation and laser-induced fluorescence analysis apparatus.

[0015] To do this, the invention provides a laser-induced fluorescenceanalysis device comprising:

[0016] a tube having a channel capable of containing a solutioncomprising at least one substance able to undergo a laser-inducedfluorescence reaction, the material of said tube being substantiallytransparent to excitation light;

[0017] at least one projection means capable of projecting an excitationlight beam locally on a portion of said channel in a direction making anangle of greater than 60° to a longitudinal direction of said channel,said excitation light being capable of inducing a fluorescence reactionin said substance or one of said substances;

[0018] at least one optical collecting means placed so as to collectfluorescence light from said channel;

[0019] at least one optical measurement means coupled to said collectingmeans so as to be able to measure said collected fluorescence light; and

[0020] a processing means capable of processing a measurement signaltransmitted by said measurement means in order to produce a result ofthe analysis of said solution;

[0021] characterized in that a first collecting means is mechanicallycoupled to a first end of said tube, said tube being made of a materialwhose refractive index is either less than that of the water in thechannel or greater than both that of the water in the channel and thatof the air surrounding the tube, so as to be able to guide saidfluorescence light along said channel in the channel and/or in the wallof the tube as far as said first collecting means, which collectingmeans is arranged so as to collect said fluorescence light propagatingapproximately along said longitudinal direction of the channel, and inthat it includes a joining means for connecting, in operation, a secondend of said tube to an output pipe of a separation system, so as toallow said solution to flow and/or said substance or substances in saidsolution to be transported between said separation system and said tube,said tube having, at said second end, an internal wall of approximatelyconical, ellipsoidal or paraboloidal shape, one face of which is turnedtoward said first end and is capable of reflecting said fluorescencelight toward said first end, said tube then having an internal crosssection larger than that of said output pipe and constituting afluorescence detection cell.

[0022] For the purpose of the invention, the water in the channelcomprises essentially water (with a refractive index of about 1.33 for awavelength of 488 nm) or water-miscible organic solvents, salts solublein water, acids commonly used for transporting substances to beseparated, or else a hydrogel (for example with a refractive index ofabout 1.36) or an electrolyte. For the purposes of the invention, thetube is capable of containing a stream or flow of liquid.

[0023] The advantage of a tube whose refractive index is less than thatof the aqueous solutions and of most common solvents is that the indexdifference at the interface between the walls of the channel, having anindex n, and the solution to be analyzed, having an index n′, generatesa high reflection coefficient, which reaches the value 1 (totalreflection) in the case of light rays with a sufficient inclination tothis interface. More precisely, if the angle of incidence of afluorescence light ray generated in the solution inside the channel isdetermined by the angle θ between this ray and the vector perpendicularto the interface pointing toward the inside of the channel, thecondition for total reflection of this ray is: θ≧arcsin(n/n′).

[0024] The tube with its channel filled with solution to be analyzedtherefore forms a waveguide with a liquid core capable of guiding thefluorescence light along its longitudinal direction with minute losses.

[0025] According to one particular feature of the invention, said tubeportion locally illuminated by said excitation light beam isapproximately adjacent to said joining means, so that said detectioncell contains the region for exciting said substance or one of saidsubstances.

[0026] Advantageously, the joining means is capable of joining the tubeto the output pipe in a rigid and substantially contiguous manner.

[0027] Advantageously, the second end of the tube has, facing away fromsaid face, an internal shoulder of cross section correspondingapproximately to the external cross section of said output pipe andserving as a stop for that part of said output pipe fitted into thetube.

[0028] Advantageously, the internal cross section of said output pipecorresponds approximately to the internal cross section of theconstriction defined between said shoulder and said face, at said secondend of the tube.

[0029] Preferably, each collecting means comprises a waveguide, a hollowT-connector being placed between said waveguide and the correspondingend of the tube in order to connect them, one in the extension of theother, a side branch of said connector allowing said solution to flowand/or said substance or substances in solution to be transported towardor from the outside of said tube.

[0030] Advantageously, in capillary electrophoresis, said lateral branchof a first T-connector connects said first end of said tube to areservoir, a first electrode being placed through a wall of saidreservoir so as to be in contact with said solution, said solution beingelectrically conducting and in contact with a second electrode placedsome distance from the first electrode, said electrodes being connectedto a voltage source so as to establish an electrical potentialdifference between the two ends of the tube in order to make saidsubstance or one of said substances migrate by electrophoresis, thewaveguide associated with said first collecting means being connectedvia said first connector to said first end in the extension of the tube.

[0031] According to one particular feature of the invention, the sidebranch of a second hollow T-connector connects said second end of saidtube to the aforementioned joining means, the waveguide associated witha second collecting means being connected via said second T-connector tosaid second end of the tube in the extension of the latter. In thiscase, said tube may also have, at said first end, an internal wall ofapproximately conical, ellipsoidal or paraboloidal shape, one face ofwhich is turned toward said second end and is capable of reflecting saidfluorescence light toward said second end.

[0032] According to another feature of the invention, the approximatelyconical (218), ellipsoidal or paraboloidal internal wall(s) of the tube(2, 202) is (are) coated with an oxidation-resistant reflective material(242).

[0033] Preferably, said tube has an internal diameter of up to 200 μmfor use in capillary electrophoresis or μ-HPLC and up to 1000 μm for usein HPLC.

[0034] Preferably, the projection means comprises a light source placedlaterally some distance from the channel and optical means placedbetween the light source and the channel in order to match the crosssection of said excitation light beam to the inside diameter of saidchannel.

[0035] Provision may be made for the external wall of the tube to becoated with a reflective material, with the exception in particular ofthe wall portion facing the excitation region of the detection cell,which must remain transparent to the beam.

[0036] The invention also provides a chromatographic or electrophoreticseparation apparatus which includes such an analysis device. In thiscase, the tube forms or is connected to a separation column of theapparatus.

[0037] The invention will be better understood, and other objects,details, features and advantages thereof will become more clearlyapparent, over the course of the following description of severalparticular embodiments of the invention, given solely by way ofillustration but implying no limitation, and with reference to theappended drawings. In these drawings:

[0038]FIG. 1 is an overall schematic view of an analysis deviceaccording to a first embodiment of the invention;

[0039]FIG. 2 is an overall schematic view of an alternative version ofthe device of FIG. 1;

[0040]FIG. 3 is an overall schematic view of an analysis deviceaccording to a second embodiment of the invention;

[0041]FIG. 4 is an enlarged view of a detail, circled at IV, of thedevice of FIG. 1; and

[0042]FIG. 5 is an enlarged view of a similar detail of the device ofFIG. 3.

[0043] In a first embodiment described with reference to FIG. 1, theanalysis device, denoted overall by 1, comprises a tube 2 made of apolymer having a refractive index less than that of water, or made ofsilica (for example with a refractive index of 1.45 for a wavelength of488 nm) . The tube 2 is a tube through which a stream of a solutioncoming from a separation apparatus 35 flows via an output pipe 3 of thisapparatus, which is for example a chromatography column, a PEEK tube ora steel tube of small cross section. The apparatus 35 is ahigh-performance liquid chromatography or micro- ornano-high-performance liquid chromatography separation apparatus of atype known to those skilled in the art and having, schematically, a mainreservoir 32 tube the dissolved substances to be separated, a separationcolumn 33 connected to the reservoir 32 and a pump 34 for making thesolution flow through the separation column 33. The separation column isconnected to the output pipe 3 or is coincident with the latter.

[0044] The tube 2 is fastened to the output pipe 3 in a rigid andcontiguous manner by an opaque connecting sleeve 4 having no deadvolume. Inside this sleeve, one end of the tube 2 butts against one endof the pipe 3. The solution contains one or more molecular substanceswhich have been separated in the separation apparatus and which it isdesired to detect and assay by fluorescence measurement.

[0045] A short portion 16 of the tube 2 is illuminated transversely andlocally by a projection means comprising an excitation light source 12emitting at least one wavelength chosen in order to induce afluorescence reaction in at least one of the substances to be detected.In order to excite the fluorescence of several substancessimultaneously, it is also possible to provide several projection meanswith excitation light sources having different wavelengths, these placedside by side along the tube 2 and/or around its periphery. In this case,the various substances will fluoresce at different wavelengths and itwill be opportune to add, downstream of the collection means, a spectralseparation system, of the optical filter, prism monochromator ordiffraction grating type, allowing one or more emission wavelength bandsto be selected. This will also be the case if the excitation source is alaser emitting in the UV, it then being possible for the varioussubstances to each undergo specific native fluorescence.

[0046] The directed beam of excitation light 13 passes through a set oflenses 14 designed to make the beam F converge on the tube portion 16 sothat the diameter of the beam F in the tube 2 is approximately equal tothe internal diameter of the channel 11 of the tube 2. Thus, the loss ofpart of the excitation light not encountered by the solution isminimized, whereas the illuminated volume of solution remains sufficientto induce fluorescence light in a detectable amount right from the verylow concentrations of fluorescent substance. The yield in convertingexcitation light into fluorescence light is therefore optimized, owingto the orthogonal geometry adopted.

[0047] The set of lenses 14 could also make the beam F diverge, forexample if the source 12 produces a narrower beam 13 than the inside ofthe tube 2. The direction of incidence of the beam F makes an angle α ofgreater than 60°, and approximately 90° in the example shown, with thedirection of the axis A of the tube 2.

[0048] Preferably, if the direction of incidence of the beam F withrespect to the axis A is inclined, it is inclined so as to reduce theangle α between said direction of incidence and the axis A beside thecollecting means 5, in order to orient the excitation light on theopposite side from the measurement means and thus reduce the excitationlight scattering at the measurement means.

[0049] When the tube has a refractive index less than that of water, thefluorescence light generated locally in the illuminated portion 16 isguided along the axial direction of the tube 2 by successive reflectionsoff its internal surface, the tube 2 forming a cylindrical wave guidewith a liquid core.

[0050]FIG. 4 shows a fluorescence light ray 27 generated at a point 28in the solution inside the channel 11, for a light ray having asufficient angle of inclination to this interface. The angle ofincidence of the ray 27 is determined by the angle θ between the ray 27and the vector 29 pointing toward the inside of the tube 2 andperpendicular to the interface 31 between the solution, having arefractive index n′, and the tube 2, having a refractive index n. Thetotal reflection condition for the ray 27 is: θ≧arcsin(n/n′). Thus, bychoosing the material of the tube 2 for minimizing the refractive indexn, the reflection coefficient at the interface 31 is high for most ofthe fluorescence light rays generated and equal to one for the rays thatsatisfy the abovementioned condition.

[0051] At the opposite end of the tube 2 from the separation system, anopaque, hollow T-connector 5 is fitted so as, on the one hand, toconnect the tube 2 to one or more collecting optical fibers 7 placedalong the extension of the tube 2 and, on the other hand, to connect thetube 2 to a discharge pipe 6 allowing the solution to flow from the tube2 perpendicular thereto. At the center of the connector 5, the tube 2butts against the collecting fiber 7 so as to minimize the loss offluorescence light by scattering at the interface between them. Thesolution to be analyzed therefore flows through the tube 2 beforere-emerging via the side branch 30 of the connector 5.

[0052] The optical fiber or fibers 7 axially collects or collect theemitted fluorescence light and guides (guide) it as far as a photomultiplier tube 8, or another type of optical detector. Thephotomultiplier tube 8 produces a measurement signal which is taken by alinking means 10 to a data processing system 9, for example amicrocomputer, which includes software means known to those skilled inthe art for processing the measurement signal received and for producingan analysis result, for example absolute or relative quantitativemeasurements of the concentration of the substance or substancesemitting fluorescence light.

[0053] The internal channel 11 of the tube 2 has, at the end fitted intothe sleeve 4, a conical, ellipsoidal or paraboloidal peripheral internalsurface in order to increase the reflection of the fluorescence light inthe direction of the collecting fibers 7, the tube then having anenlarged internal cross section compared with that of the pipe 3.

[0054] Because of the good optical transmission between the tube 2 andthe collecting fiber 7, it is unnecessary for there to be spectral orspatial filtering means between the tube 2 and the optical detector,although it is also possible to provide such means in order to improvethe detection thresholds. Although not shown, electrical supply meansare incorporated into or connected to the photomultiplier tube 8 and tothe light source 12 in order to operate them.

[0055] In an alternative version of the first embodiment, shown in FIG.2, the analysis device 101 includes, in place of the connecting sleeve4, a second opaque hollow T-connector 105, identical or similar to theT-connector 5, which joins the tube 2, via a right-angled side branch130 connected to a joining means 104, to the output pipe 103, similar tothe pipe 3, and connects a second bundle 107 of collecting opticalfibers, identical or similar to the fibers 7, lying along the extensionof the tube 2, in order to guide fluorescence light as far as a secondphotomultiplier tube 108, identical or similar to the photomultipliertube 8. The photomultiplier tube 108 may be connected via a line 110 tothe aforementioned system 9. In this version, the arrangement at the twoends of the tube 2 is therefore symmetrical so as to collect all thefluorescence light trapped in the tube 2, whatever its direction ofpropagation. The solution enters the tube 2 via the pipe 103 and leavesvia the discharge pipe 6. The other parts of the device 101 areidentical or similar to those of the device 1 of FIG. 1. In this case,one end or both ends (as shown in FIG. 2) of the tube has or have aconical, ellipsoidal or paraboloidal wall.

[0056] A second embodiment of the invention will now be described withreference to FIG. 3, in which the elements identical or similar to thoseof the first embodiment bear the same reference numbers as in FIG. 1,increased by 200. The analysis device 201 is connected, in operation, toan electrophoretic separation system 235, for example one operating bycapillary electrophoresis, isotachophoresis or electro-chromatography,the output pipe 203 of which is a silica capillary or an electrophoreticseparation tube. The solution is an electrolyte contained in the mainreservoir 232 of the separation system 235, the pipe 203 connected tothe reservoir 232 via a column 233, the tube 202 connected to the pipe203, the inside of the T-connector 205 and a lateral buffer reservoir206. The electrolyte is in electrical contact with a first electrode 224of the separation system, immersed in the reservoir 232, and with asecond electrode 217 inserted in a sealed manner through the wall of thebuffer reservoir 206. For example, the reservoir 206 is in the form of atest tube, the open end of which is sealed into the side branch 230 ofthe connector 205 and the electrode 217 passes in a sealed mannerthrough the bottom of said test tube. Of course, the reservoir 206 issubjected to atmospheric pressure via an appropriate aperture. There isthus electrical continuity parallel to the axis of the pipe 203 and ofthe tube 202 and, at the T-connector 205, perpendicular to the axis ofthe tube 202.

[0057] In operation, these electrodes are connected to a DC voltagesource 226 of the separation system 235 allowing a potential drop to beestablished within the pipe 203 and the tube 202 for separatingdissolved substances by electrophoresis. The junction 204 between thetube 202 and the pipe 203 is obtained by the latter fitting into theformer, the inside diameter of the tube 202 being coincident with theoutside diameter of the pipe 203. At this junction, the internal wall ofthe tube 202 has a frustoconical, or ellipsoidal or paraboloidalconstriction whose face 218 allows the fluorescence light to bereflected toward the collecting fibers 207 so as to substantiallyincrease the efficiency of collection of fluorescence light by thefibers 207. Greater performance may be obtained if the face 218 iscoated with an oxidation-resistant reflective material 242, such asgold, silver or platinum, for example by thin-film deposition by vacuumevaporation (see FIG. 5). This constriction has, facing away from theinclined face 218, a shoulder 219 serving as a stop for that part of thepipe 203 fitted into the tube 202. To prevent the accumulation ofcontaminants at the junction, the interface between the tube 202 and thepipe 203 must be very uniform. This is obtained by having a good surfacefinish and a tight fit with adhesive bonding.

[0058] The operation of the analysis device 201 is similar to that ofthe device 1. The light source 212 and the set of lenses 214 direct theexcitation light beam 213 on to a portion 216 of the tube 202. Theportion 216 subjected to the local excitation illumination is preferablychosen to be adjacent to the frustoconical constriction and to thejunction with the pipe 203, so as to reduce the dead volume and theinfluence of the variation in internal diameter of the separation tubeformed by the pipe 203 and the tube 202. The photometric detector 208transmits a measurement signal via the linking means 210 to theprocessing system 209.

[0059] The tube 2 or 202 has a large internal diameter, for example upto 200 μm for use in capillary electrophoresis or μ-HPLC and up to 1000μm for use in HPLC, so as to optimize the illuminated volume of solutionand, consequently, the fluorescence detection sensitivity.

[0060] The cylindrical shape of a capillary channel favors thepropagation of the fluorescence emitted inside the capillary tube,provided that the refractive index of the propagation channel is higherthan the index of the “outer ” adjacent medium.

[0061] Propagation therefore takes place in the liquid stream containedin the capillary channel 2 if the latter is made of a polymer having anindex less than that of water or of the commonly used solvents.

[0062]FIG. 5 shows a silica tube 202, having a refractive index ngreater than that of water, with a fluorescence light ray 227 generatedat a point 228 in the solution inside the channel 211. The angle ofincidence of the ray 227 is determined by the angle θ between the ray227 and the vector 229 pointing toward the inside of the tube 202 andperpendicular to the interface 231 between the solution, having arefractive index n′, and the wall of the tube 202, having a refractiveindex n. Since n is greater than n′, there will be, of course, totalreflection of the ray 227 which propagates along the silica wall. Sincethis index n is greater than that n″ of the air surrounding the tube202, depending on the aforementioned angle of incidence and the chosenvalue of n, the ray is at least partly reflected, at the interfacebetween the tube and the surrounding air, toward the inside of the walland into the channel. Preferably, the index n″is less than the index n.No sleeve is necessary owing to the fact that the light is collectedalong the longitudinal axis.

[0063] As can be seen in FIGS. 4 and 5, one propagation direction ispreferential here: for this, the capillary tube has an enlarged crosssection constituting a detection cell 216 adjacent to the joining means204. The enlarged region of conical, ellipsoidal or paraboloidal shapemay also be coated, on the inside, with a reflective material, made ofgold, silver or platinum, by a process such as thin-film deposition byvacuum evaporation. This widening of the capillary tube 202 at theexcitation region 216 allows a larger sample volume to be irradiated.

[0064] The addition of a coating 243 acting as a mirror on the externalwall of the capillary tube 202 makes it possible to reduce the criticalangle θ (the angle relative to the vector 229) and thus decrease thefluorescence transmitted/scattered in the air surrounding the tube.However, this coating 243 must not cover the excitation region 216 ofthe detection cell, which must remain transparent to the beam F.

[0065] It is also possible to use a sleeve that has the same externalreflective coating 243, into which sleeve are inserted and adjusted theseparation capillary tube 203 and the wider capillary tube 202 servingas detection cell, preferably with adhesive bonding, thereby allowingthis sleeve to be machined to the desired shape.

[0066] In order to maintain the electrophoretic and chromatographicresolutions, the excitation beam F may have a cross section ofelliptical shape, the major axis of this ellipse being perpendicular tothe longitudinal axis A of the capillary tube and the minor axis of thisellipse being parallel to said axis A.

[0067] Of course, the tube 2 may be made of silica and, conversely, thetube 212 may be made of a polymer without departing from the scope ofthe invention.

[0068] Although the invention has been described in relation to severalparticular embodiments, it is obvious that it is in no way limitedthereby and that it comprises all technical equivalents of the meansdescribed and also their combinations, provided that these fall withinthe scope of the invention.

1. A laser-induced fluorescence analysis device (1, 101, 201)comprising: a tube (2, 202) having a channel (11, 211) capable ofcontaining a solution comprising at least one substance able to undergoa laser-induced fluorescence reaction, the material of said tube beingsubstantially transparent to excitation light; at least one projectionmeans (12, 14; 214, 212) capable of projecting an excitation light beam(13, 213) locally on a portion (16, 216) of said channel in a directionmaking an angle of greater than 60° to a longitudinal direction (A) ofsaid channel, said excitation light being capable of inducing afluorescence reaction in said substance or one of said substances; atleast one optical collecting means (7, 107, 207) placed so as to collectfluorescence light from said channel; at least one optical measurementmeans (8, 108, 208) coupled to said collecting means so as to be able tomeasure said collected fluorescence light; and a processing means (9,209) capable of processing a measurement signal transmitted by saidmeasurement means in order to produce a result of the analysis of saidsolution; characterized in that a first collecting means (7, 207) ismechanically coupled to a first end of said tube (2, 202), said tubebeing made of a material whose refractive index is either less than thatof the water in the channel (11, 211) or greater than both that of thewater in the channel and that of the air surrounding the tube (2, 202),so as to be able to guide said fluorescence light along said channel inthe channel and/or in the wall of the tube as far as said firstcollecting means, which collecting means is arranged so as to collectsaid fluorescence light propagating approximately along saidlongitudinal direction of the channel, and in that it includes a joiningmeans (4, 104, 204) for connecting, in operation, a second end of saidtube to an output pipe (3, 103, 203) of a separation system, so as toallow said solution to flow and/or said substance or substances in saidsolution to be transported between said separation system and said tube,said tube (2, 202) having, at said second end, an internal wall (218) ofapproximately conical, ellipsoidal or paraboloidal shape, one face ofwhich is turned toward said first end and is capable of reflecting saidfluorescence light toward said first end, said tube then having aninternal cross section larger than that of said output pipe (3, 203) andconstituting a fluorescence detection cell (16, 216).
 2. The device asclaimed in claim 1, characterized in that said tube portion (16, 216)locally illuminated by said excitation light beam is approximatelyadjacent to said joining means, so that said detection cell contains theregion for exciting said substance or one of said substances.
 3. Thedevice as claimed in claim 1, characterized in that the second end ofthe tube has, facing away from said face (218), an internal shoulder(219) of cross section corresponding approximately to the external crosssection of said output pipe and serving as a stop for that part of saidoutput pipe (203) fitted into the tube (202).
 4. The device as claimedin claim 3, characterized in that the internal cross section of saidoutput pipe corresponds approximately to the internal cross section ofthe constriction defined between said shoulder and said face, at saidsecond end of the tube.
 5. The device as claimed in claim 1,characterized in that each collecting means comprises a waveguide (7,107, 207), a hollow T-connector (5, 105, 205) being placed between saidwaveguide and the corresponding end of the tube in order to connectthem, one in the extension of the other, a side branch (30, 130, 230) ofsaid connector allowing said solution to flow and/or said substance orsubstances in solution to be transported toward or from the outside ofsaid tube.
 6. The device as claimed in claim 5, characterized in thatsaid lateral branch (230) of a first T-connector (205) connects saidfirst end of said tube to a reservoir (206), a first electrode (217)being placed through a wall of said reservoir so as to be in contactwith said solution, said solution being electrically conducting and incontact with a second electrode (224) placed some distance from thefirst electrode, said electrodes being connected to a voltage source(226) so as to establish an electrical potential difference between thetwo ends of the tube (202) in order to make said substance or one ofsaid substances migrate by electrophoresis, the waveguide (207)associated with said first collecting means being connected via saidfirst connector to said first end in the extension of the tube.
 7. Thedevice as claimed in claim 5, characterized in that the side branch(130) of a second hollow T-connector (105) connects said second end ofsaid tube to the aforementioned joining means (104), the waveguide (107)associated with a second collecting means being connected via saidsecond T-connector to said second end of the tube in the extension ofthe latter.
 8. The device as claimed in claim 7, characterized in thatsaid tube (2) also has, at said first end, an internal wall ofapproximately conical, ellipsoidal or paraboloidal shape, one face ofwhich is turned toward said second end and is capable of reflecting saidfluorescence light toward said second end.
 9. The device as claimed inclaim 1, characterized in that the approximately conical (218),ellipsoidal or paraboloidal internal wall(s) of the tube (2, 202) is(are) coated with an oxidation-resistant reflective material (242). 10.The device as claimed in claim 1, characterized in that said tube (2,202) has an internal diameter of up to 200 μm for use in capillaryelectrophoresis or μ-HPLC and up to 1000 μm for use in HPLC.
 11. Thedevice as claimed in claim 1, characterized in that said projectionmeans comprises a light source (12, 212) placed laterally some distancefrom said channel and optical means (14, 214) placed between the lightsource and the channel in order to match the cross section of saidexcitation light beam to the inside diameter of said channel.
 12. Thedevice as claimed in claim 11, characterized in that the external wallof the tube is coated with a reflective material (243), with theexception in particular of the wall portion facing the excitation region(216) of the detection cell, which must remain transparent to the beam(F).
 13. The device as claimed in claim 1, characterized in that theexcitation beam (F) has a cross section of elliptical shape, the majoraxis of this ellipse being perpendicular to the longitudinal axis (A) ofthe capillary tube and the minor axis of this ellipse being parallel tosaid longitudinal axis (A) of the tube.
 14. A chromatographic orelectrophoretic separation apparatus (35, 235), characterized in that itincludes an analysis device (1, 101, 201) as claimed in claim
 1. 15. Thedevice as claimed in claim 2, characterized in that the second end ofthe tube has, facing away from said face (218), an internal shoulder(219) of cross section corresponding approximately to the external crosssection of said output pipe and serving as a stop for that part of saidoutput pipe (203) fitted into the tube (202).
 16. The device as claimedin claim 6, characterized in that the side branch (130) of a secondhollow T-connector (105) connects said second end of said tube to theaforementioned joining means (104), the waveguide (107) associated witha second collecting means being connected via said second T-connector tosaid second end of the tube in the extension of the latter.